! Project Summary/Abstract Fundamental analysis of the mechanisms of leukemogenesis is required in order to develop optimized treatment approaches. N6-methyladenosine (m6A), the most abundant RNA modification, plays key roles in RNA metabolism, transcript stability, and translation efficiency. Aberrant regulation of m6A writers and erasers contributes to leukemogenesis, though the exact mechanisms how the methyltransferase machinery is altered in leukemia remains to be shown. Mutations in methyltransferase writers have not been identified to date, with one exception: in the recurrent t(1;22) translocation in acute megakaryoblastic leukemia (AMKL) a component of the m6A writer machinery, RBM15, is fused with MKL1, a transcriptional cofactor of serum response factor (SRF). Understanding the mechanism by which the RBM15-MKL1 (RM) fusion protein causes leukemia will shed light on the role of disordered m6A RNA methylation in leukemogenesis, specifically AMKL but also AML and to cancer in general. We hypothesize that for RM-associated AMKL to develop, the MKL1 domain aberrantly targets RBM15 activity to sites of SRF binding and that RBM15 recruits the m6A writer complex to associated RNAs, altering m6A mRNA methylation and expression of genes that are required for transformation and/or are important for megakaryocyte fate commitment and maturation. We propose focused, unbiased genome-wide studies to determine how the m6A epitranscriptome and the SRF transcriptional networks are coopted in RBM15- MKL1 AMKL. The functional effects of candidate target genes common to the genomic approaches (m6A RNA immunoprecipitation, chromatin mapping, RNA stability assays, and mapping of the translatome) as well as the relevance of critical domains in RM-mediated leukemogenesis will be tested with assays of growth, differentiation and oncogene dependence in RM-induced murine AMKL as well as primary human AMKL patient derived xenotransplants (PDX). The studies are highly clinically relevant as they address a unique mechanism causative of AMKL via comprehensive analysis of fundamental biologic mechanisms, and will reveal previously unidentified regulation of the epitranscriptome that may be a novel shared oncogenic mechanism in AML and other cancers. The proposed approaches are multifaceted, using cell lines as well as genetically engineered animal models, and primary human and murine leukemia samples. The studies will contribute to our understanding of leukemogenesis by elucidating the direct role of a component of the m6A RNA methylase complex in leukemia shedding light on the broader role of m6A mRNA modifications in AML. !